Thrombin Substrate and Assay for Determining the Level of Bioactive Thrombin in a Sample

ABSTRACT

Substrates for thrombin and assays for determining the level of bioactive thrombin in a sample are disclosed, wherein the substrate has the general formula: A-X-Z-A′ wherein one of either A or A′ comprises a luminescent chelate and the other one of A or A′ comprises a first partner of a binding pair, X forms a tri- or tetra-peptide, and Z comprises a linker.

FIELD OF THE INVENTION

The present invention relates to novel substrates for thrombin andassays for determining the level of bioactive thrombin in a sample.

BACKGROUND OF THE INVENTION

Thrombin is produced by the enzymatic cleavage of two sites onprothrombin by activated Factor X (Xa).

Thrombin converts fibrinogen to an active form that assembles intofibrin. Thrombin also activates Factor XI, Factor V and Factor VIII.This positive feedback accelerates the production of thrombin.

Factor XIII is also activated by thrombin. Factor XIIIa is atransglutaminase that catalyses the formation of covalent bonds betweenlysine and glutamine residues in fibrin. The covalent bonds increase thestability of the fibrin clot.

In addition to its activity in the coagulation cascade, thrombin alsopromotes platelet activation, via activation of protease-activatedreceptors on the platelet.

In clinical practice the measurement of prothrombin time (PT), i.e., thetime it takes for blood to clot, in terms of activated partialthromboplastin time (APTT) and activated clotting time (ACT), is widelyused for screening for defects of coagulation pathways and formonitoring anticoagulant therapy.

In order to standardize the results of blood coagulation tests, theconcept of International Normalized Ratio (INR) has been devised. Eachmanufacturer of tissue factors gives an ISI (International SensitivityIndex) for any tissue factor they make. The ISI value indicates thecomparison between a particular batch of tissue factor and aninternationally standardized sample. As depicted below, INR is the ratioof a patient's prothrombin time to a control sample's prothrombin time,raised to the power of the ISI value for the thromboplastin reagentused.

INR=(PT _(test) /PT _(normal))_(ISI)

There are two kinds of assays available for the measurement of PT. Onekind of assay is coagulometric, which is based on the endpoint of clotformation from fibrinogen to fibrin conversion. The results of theassays are, however, variable, and particularly affected by interferencewith the fibrinogen/fibrin conversion in patients. Another kind of assayis non-coagulometric and is based on the use of synthetic substratessuitable for thrombin cleavage. The results of the latter assays areless variable and not affected by the fibrinogen level.

U.S. Pat. No. 4,061,625 discloses chromogenic thrombin substrates of theformula

D-Phe-cyclic imino acid-Arg-pNA

wherein the cyclic imino acid is selected from among 2-azetidinecarboxylic acid, proline, 2-piperidine carboxylic acid, and pNA isp-nitroanilide. These substrates are described as suitable for aquantitative determination of thrombin or for a study of reactions inwhich thrombin is formed, inhibited or consumed, or for determination offactors which exert an influence or take part in such reactions e.g.,for determination of anti-thrombin, prothrombin and heparin.

When chromogenic substrates are used, the reaction is detected atapproximately 405 nm. At this wavelength, whole blood can not beanalysed, due to the absorption from the whole blood in its self.Chromogenic substrates are therefore not suitable when the used sampletype is whole blood.

Time-resolved luminescence spectroscopy using chelates such aslanthanide chelates has for several years been applied in immunoassaysand DNA hybridization assays. Due to the absorption properties of wholeblood, this chelate detection technology is suitable when the sample iswhole blood, compared to the chromogenic substrates, because it ispossible with this technology to measure absorbance at 615 nm, at whichwavelength, whole blood absorption is low.

Such chelates are described in U.S. Pat. No. 7,018,851 B1 whichdiscloses improved fluorescent lanthanide chelates which are suitablefor time-resolved fluorometric (TRF) applications. The chelates are usedin specific bioaffinity based binding assays such as immunoassays.

TRF is a suitable detection technology for assays requiring highsensitivity and wide dynamic range. In immunoassay techniques usingconventional fluorescence detection, high non-specific background causedby light scattering, e.g., from the biological components of the sampleis a severe limitation to the sensitivity of the assay.

The fluorescent lanthanide chelates have traditionally been used inimmunoassays where the detection principle is based on the capture ofsuch chelates and detection thereof. In contrast they have not been usedin enzyme assays, where the detection principle is based on cleavage ofthe chelate substrate and detection of the captured (not cleaved)substrate remaining in the assay.

The key feature of such a system is the provision of a substrate whichis on the one hand stable and on the other hand specifically cleavableby thrombin.

Therefore, there exists a need for an enzyme substrate which isspecifically cleavable by thrombin and which comprises an intrinsicstable luminescent component having a long lifetime, thus facilitatingdetection in whole blood and resulting in less interference.

Furthermore there exists a need for an assay which is fast, easy toperform, subject to low variability, easily automated, and of low cost.

Surprisingly it has been found that by combining, via a certain linker,a peptide which is specifically cleavable by thrombin, with aluminescent chelate, a stable substrate with a long lifetime, which canbe used when the sample type is whole blood, is provided.

SUMMARY OF THE INVENTION

In a first aspect, the present invention is a substrate for thrombinhaving the formula:

A-X-Z-A′

whereinone of either A or A′ comprises a luminescent chelate, andthe other one of A or A′ comprises a first partner of a binding pair,optionally including a spacer, and connected via a peptide bond to theremaining part of the substrate,X forms a tri- or tetra-peptide selected from among X′-Phe-Aze-Arg,X′-Phe-Pip-Arg, and X′-Phe-Pro-Arg, whereinX′ is absent or selected among Lys, Ahx, Ile, and Val,Z is NH—R—Z′, whereinR is selected from the group consisting of C₁₋₆alkylene, C₁₋₆alkylenoxy,C₁₋₆thioalkylene, C₁₋₆thioalkylenoxy, carbonyl-C₁₋₆alkylene,carbonyl-C₁₋₆alkylenoxy, C₁₋₆alkylene-carbonyl, C₁₋₆alkylenoxy-carbonyl,C₁₋₆alkylene-arylene, C₁₋₆alkylenoxy-arylene, C₁₋₆alkylene-NH,C₁₋₆alkylenoxy-NH, C₁₋₆alkylene-NHCO, C₁₋₆alkylenoxy-NHCO,C₁₋₆alkylene-CONH, C₁₋₆alkylenoxy-CONH, C₁₋₆alkylene-COS,C₁₋₆alkylenoxy-COS, C₁₋₆alkylene-CONH—C₁₋₆alkylene-arylene, arylene,arylene-C₁₋₆alkylene, arylene-C₁₋₆alkylenoxy, R¹_(a)-arylene-(NHCO—R²)_(b), R³ _(c)-arylene-(CONH—R⁴)_(d),(R⁵—CONH)_(e)-arylene-R⁶ _(f), and (R⁷—NHCO)_(g)-arylene-R⁸ _(h),wherein each R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ at each occurrence isindependently selected from among C₁₋₆alkylene, C₁₋₆alkylenoxy,C₁₋₆thioalkylene, C₁₋₆thioalkylenoxy, carbonyl-C₁₋₆alkylene,carbonyl-C₁₋₆alkylenoxy, C₁₋₆alkylene-carbonyl, arylene andarylene-C₁₋₆alkylene, and wherein each of a, b, c, d, e, f, g, and h isindependently selected from among the integers from 0 to 6,wherein the arylene is phenylene, biphenylene or naphthylene, whichphenylene, biphenylene or naphthylene is optionally mono-, di- ortri-substituted by one or more substituents selected from among halogen,OH, SH, CN, NO₂, C₁₋₆alkyl, C₁₋₆alkoxy, and C₁₋₆alkoxycarbonyl,Z′ comprises one of N, S, and carbonyl, wherein,when Z′ comprises N, Z′ is selected from among thiourea (—NH—CS—NH—),aminoacetamide (—NH—CO—CH₂—NH—), amide (—NH—CO—), methylamide(—NCH₃—CO—) and substituted-triazine-diamine (—NH—(R⁹C₃N₃)—NH—),when Z′ comprises S, Z′ is selected from among thioether (—S—),thioacetamide (—S—CH₂—CO—NH—), disulfide (—S—S—), (—S—CO—CH₂—NH—) and(—S—(R⁹C₃N₃)—NH—) orwhen Z′ comprises carbonyl, Z′ is selected from among an amide (—CO—NH—,—CO—NCH₃—) and an ester (—CO—O—),wherein R⁹ is selected from the group consisting of hydrogen, halogen,C₁₋₆alkyl, C₁₋₆thioalkyl, C₁₋₆alkoxy, C₁₋₆thioalkoxy, aryloxy, andamino, which alkyl, thioalkyl, alkoxy, thioalkoxy or aryloxy group isoptionally mono-, di- or tri-substituted and which amino group isoptionally mono- or di-substituted by one or more substituents selectedfrom among halogen, OH, SH, CN, NO₂, C₁₋₆alkyl, C₁₋₆alkoxy, andC₁₋₆alkoxycarbonyl.

Another aspect of the invention is a one-step assay method fordetermining the level of bioactive thrombin in a test sample comprisingthe steps:

-   a) combining, sequentially, simultaneously or substantially    simultaneously, a substrate according to claim 1, an activator and    said test sample;-   b) incubating the resulting reaction mixture to release    thrombin-cleaved, chelate-containing substrate fragments and    immobilising the binding partner-containing substrate fragment and    non-thrombin-cleaved intact substrate on an immobilisation matrix;-   c) washing off non-immobilised, thrombin-cleaved chelate-containing    substrate fragment and non-immobilised, non-thrombin-cleaved    substrate, if present;-   d) measuring the level of luminescent emission from immobilized    intact substrate; and-   e) calculating thrombin activity from the reduction of intensity of    luminescent emission compared to a thrombin-free standard sample.

Another aspect of the invention is a two-step assay method fordetermining the level of bioactive thrombin in a test sample comprisingthe steps:

-   a) adding said sample and an activator to a substrate according to    claim 1 in liquid phase,-   b) incubating the resulting reaction mixture to release    thrombin-cleaved, chelate-containing substrate fragment,-   c) adding the reaction mixture to an immobilisation matrix,-   d) washing off non-immobilised, thrombin-cleaved chelate-containing    substrate fragment and non-immobilised, non-thrombin-cleaved    substrate, if present,-   e) measuring the level of luminescent emission from immobilised    intact substrate, and-   f) calculating thrombin activity from the reduction of intensity of    luminescent emission compared to a thrombin-free standard sample.

Another aspect of the invention is a test kit for determining the levelof bioactive thrombin in a sample comprising

-   a) a luminescent substrate according to claim 1, and-   b) a solid immobilisation matrix.

Another aspect of the invention is a substrate for an enzyme having theformula:

A-X-Z-A′

whereinone of either A or A′ comprises a luminescent chelate, andthe other one of A or A′ comprises a first partner of a binding pair,optionally including a spacer, and connected via a peptide bond to theremaining part of the substrate,X forms a tri- or tetra-peptide,Z is NH—R—Z′, wherein

R is selected from the group consisting of C₁₋₆alkylene, C₁₋₆alkylenoxy,C₁₋₆thioalkylene, C₁₋₆thioalkylenoxy, carbonyl-C₁₋₆alkylene,carbonyl-C₁₋₆alkylenoxy, C₁₋₆alkylene-carbonyl, C₁₋₆alkylenoxy-carbonyl,C₁₋₆alkylene-arylene, C₁₋₆alkylenoxy-arylene, C₁₋₆alkylene-NH,C₁₋₆alkylenoxy-NH, C₁₋₆alkylene-NHCO, C₁₋₆alkylenoxy-NHCO,C₁₋₆alkylene-CONH, C₁₋₆alkylenoxy-CONH, C₁₋₆alkylene-COS,C₁₋₆alkylenoxy-COS, C₁₋₆alkylene-CONH—C₁₋₆alkylene-arylene, arylene,arylene-C₁₋₆alkylene, arylene-C₁₋₆alkylenoxy, R¹_(a)arylene-(NHCO—R²)_(b), R³ _(c)-arylene-(CONH—R⁴)_(d),(R⁵—CONH)_(e)-arylene-R⁶ _(f), and (R⁷—NHCO)_(g)-arylene-R⁸ _(h),

wherein each R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ at each occurrence isindependently selected from among C₁₋₆alkylene, C₁₋₆alkylenoxy,C₁₋₆thioalkylene, C₁₋₆thioalkylenoxy, carbonyl-C₁₋₆alkylene,carbonyl-C₁₋₆alkylenoxy, C₁₋₆alkylene-carbonyl, arylene andarylene-C₁₋₆alkylene, and each of a, b, c, d, e, f, g, and h isindependently selected from among the integers from 0 to 6,wherein the arylene is phenylene, biphenylene or naphthylene, whichphenylene, biphenylene or naphthylene is optionally mono-, di- ortri-substituted by one or more substituents selected from among halogen,OH, SH, CN, NO₂, C₁₋₆alkyl, C₁₋₆alkoxy, and C₁₋₆alkoxycarbonyl,Z′ comprises one of N, S, and carbonyl, wherein,when Z′ comprises N, Z′ is selected from among thiourea (—NH—CS—NH—),aminoacetamide (—NH—CO—CH₂—NH—), amide (—NH—CO—), methylamide(—NCH₃—CO—) and substituted-triazine-diamine (—NH—(R⁹C₃N₃)—NH—),when Z′ comprises S, Z′ is selected from among thioether (—S—),thioacetamide (—S—CH₂—CO—NH—), disulfide (—S—S—), (—S—CO—CH₂—NH—) and(—S—(R⁹C₃N₃)—NH) or when Z′ comprises carbonyl, Z′ is selected fromamong an amide (—CO—NH—, —CONCH_(S)—) and an ester (—CO—O—),

wherein R⁹ is selected from the group consisting of hydrogen, halogen,C₁₋₆alkyl, C₁₋₆thioalkyl, C₁₋₆alkoxy, C₁₋₆thioalkoxy, aryloxy, andamino, which alkyl, thioalkyl, alkoxy, thioalkoxy or aryloxy group isoptionally mono-, di- or tri-substituted and which amino group isoptionally mono- or di-substituted, by one or more substituents selectedfrom among halogen, OH, SH, CN, NO₂, C₁₋₆alkyl, C₁₋₆alkoxy, andC₁₋₆alkoxycarbonyl.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is disclosed in more detail by reference to the drawings,although it is understood that the drawings merely represent specificembodiments of the described invention and are not intended to so limitthe invention.

FIG. 1 shows a schematic of a one-step assay method without priorsubstrate immobilisation.

FIG. 2 shows a schematic of a one-step assay method with prior substrateimmobilisation.

FIG. 3 shows a schematic of a two-step assay method.

FIG. 4 shows the reaction scheme of the synthesis of S1V6c, a substrateaccording to the invention.

FIG. 5 shows the reaction scheme of the synthesis of S1V12a, anothersubstrate according to the invention.

FIG. 6 shows a standard curve of S1V6c and S1v9b according to theinvention.

DETAILED DESCRIPTION

In the context of this invention a C₁₋₆alkyl group means a straightchain or branched chain group of one to six carbon atoms, including butnot limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,t-butyl, pentyl, and hexyl. In exemplary embodiments, the C₁₋₆alkylgroup is methyl, ethyl, propyl or isopropyl.

In the context of this invention a C₁₋₆alkylene radical means a straightchain or branched chain radical of one to six carbon atoms, includingbut not limited to, methylene, ethylene, propylene, isopropylene,butylene, isobutylene, t-butylene, pentylene, and hexylene. In exemplaryembodiments, the C₁₋₆alkylene group is methylene, ethylene, propylene orisopropylene.

In the context of this invention a C₁₋₆alkoxy group means a straight orbranched chain group of one to six carbon atoms linked to an oxygenatom, including but not limited to, methoxy, ethoxy, propoxy,isopropoxy, butoxy, isobutoxy, t-butoxy, pentoxy, and hexoxy. Inexemplary embodiments, the C₁₋₆alkoxy group is methoxy, ethoxy, orpropoxy.

In the context of this invention a C₁₋₆alkylenoxy radical means astraight or branched chain radical of one to six carbon atoms linked toan oxygen atom, including but not limited to, methylenoxy, ethylenoxy,propylenoxy, isopropylenoxy, butylenoxy, isobutylenoxy, t-butylenoxy,pentylenoxy, and hexylenoxy. In exemplary embodiments, theC₁₋₆alkylenoxy group is methylenoxy, ethylenoxy, or propylenoxy.

Amino acid residues in the context of this invention have theirconventional three-letter abbreviations. Thus, Ahx stands for 2-aminohexanoic acid (norleucine), Ala stands for alanine, Arg stands forarginine, Asn stands for asparagine, Asp stands for aspartic acid, Azestands for azetidine-2-carboxylic acid, Cys stands for cysteine, Glnstands for glutamine, Glu stands for glutamic acid, Gly stands forglycine, His stands for histidine, Ile stands for isoleucine, Leu standsfor leucine, Lys stands for lysine, Met stands for methionine, Phestands for phenylalanine, Pip stands for piperidine-2-carboxylic acid,Pro stands for proline, Ser stands for serine, Thr stands for threonine,Trp stands for tryptophane, Tyr stands for tyrosine, and Val stands forvaline.

In the context of this invention halogen represents fluoro, chloro,bromo or iodo.

The substrate according to the invention must be able to be attached toan immobilisation matrix in order to separate cleaved fromnon-cleaved/intact substrates. Thus the substrate according to theinvention is terminated by a first partner of a binding pair, which inturn binds to a second partner of the binding pair. This second partnerof said binding pair is immobilised on an immobilisation matrix.

The substrate according to the invention can be quickly cleaved bythrombin with excellent specificity. Its central part is a smallpeptide, in terms of a tri- or tetra-peptide, which is cleaved bythrombin at the scissile bond located between the Arg-moiety and theZ-moiety.

The substrate according to the invention can be used when the sample is,for example, plasma and is particularly advantageous as it is suitablewhen the sample is, for example, whole blood, because the substrate, dueto the chelate technology employed, can be detected at 615 nm.

In an embodiment according to the invention, the substrate is one,wherein X′ is absent, Phe is D-Phe, and Arg is L-Arg.

In an embodiment of the invention, X is D-Phe-L-2-Aze-L-Arg orD-Phe-L-2-Pip-L-Arg.

These substrates have shown to provide rapid cleavage kinetics and/orthey are highly specific for thrombin.

In an embodiment of the invention, Z′ is N and R is arylene,C₁₋₆alkylene-arylene, or R¹ _(a)-arylene-(NHCO—R²)_(b), wherein R¹ andR² are as defined above and a and b are independently 0, 1 or 2.

Thus, substrates, wherein Z at the scissile bond comprises an aromaticmoiety, have shown to provide excellent and rapid cleavage kinetics.

In an embodiment of the invention, Z′ is a6-substituted-1,3,5-triazine-2,4-diamine (NH—(R⁹C₃N₃)—NH) or(S—(R⁹C₃N₃)—NH) bond, wherein R⁹ is selected from the group consistingof chloro, fluoro, ethoxy, 2-methoxyethoxy, 2-cyanoethoxy,2,2,2-trifluoroethoxy, thiophenoxy and ethoxycarbonylthiomethoxy.

Substrates according to this embodiment of the invention are especiallyspecific for thrombin.

In an embodiment according to the invention, the luminescent chelate isa fluorescent lanthanide chelate e.g., as disclosed in U.S. Pat. No.7,018,851 B2. Such luminescent lanthanide chelates provide highabsorptivity at a suitable wavelength, several separate UV absorbingparts in the same ligand structure, effective energy transfer from theUV absorbing part to the lanthanide ion, a strongly chelating part tocreate thermodynamic stability and high kinetic stability, and afunctional group allowing effective coupling of the chelate to be usedas a binding reactant without destroying its binding properties, andthus are suitable for the present invention.

In an exemplary embodiment according to the invention, the lanthanidechelates A or A′ are selected from:

Further, such chelates may include, for example, but are not limited to,stable chelates composed of derivatives of pyridine; bipyridines,terpyridines and various phenolic compounds as the energy mediatinggroups and polycarboxylic acids as chelating parts. In addition, variousdicarboxylate derivatives, macrocyclic cryptates, calixarenes, DTPAcarbostril 124 conjugate and macrocyclic Schiff bases have beendisclosed in patent applications and/or patents.

In an embodiment of the invention, the binding pair is selected amongbiotin as the first partner and streptavidin as the second partner(biotin/streptavidin). In other particular embodiments, the binding pairis selected from biotin/avidin, biotin/biotin acceptor peptide, andstreptavidin derivative/acceptor peptide.

The above list of binding pairs is non-exhaustive. Generally, thebinding pair comprises any binding pair capable of immobilising thesubstrate according to the invention to an immobilisation matrix.Examples of commercially available binding pairs include, but are notlimited to, Strep-tag or Strep-tagII/Strep-Tactin or Biotin/Biotinacceptor peptide.

In a particular embodiment, the first partner is biotin.

In a particular embodiment, the second partner is streptavidin.

Immobilisation matrixes are known to a person skilled in the art andinclude micro wells, micro particles such as beads, e.g., those made ofglass or plastic materials such as, but not limited to, polystyrene orpolypropylene.

In an embodiment according to the invention the first partner of abinding pair is connected to the remaining part of the substrate via aspacer. A spacer can result in an improved solubility of the substrate,which is particularly useful for steric reasons. Thus the role of thespacer is to solubilise the peptide/linker/chelate part of the substratein order to reduce steric hindrance otherwise imposed on the thrombinenzyme.

In an embodiment according to the invention, the spacer is of theformula NH—R¹⁰—CONH—R¹¹—CO—(NH—R¹²—NH)_(i), wherein each of R¹⁰, R¹¹ andR¹² is independently selected from among C₁₋₆alkylene, C₁₋₆alkylenoxy,C₁₋₆thioalkylene, C₁₋₆thioalkylenoxy, carbonyl-C₁₋₆alkylene,carbonyl-C₁₋₆alkylenoxy, C₁₋₆alkylene-carbonyl, arylene andarylene-C₁₋₆alkylene, wherein arylene is phenylene, biphenylene ornaphthylene, which phenylene, biphenylene or naphthylene is optionallymono-, di- or tri-substituted by one or more substituents selected fromamong halogen, OH, SH, CN, NO₂, C₁₋₆alkyl, C₁₋₆alkoxy andC₁₋₆alkoxycarbonyl, wherein i is an integer from 0 to 6.

In an embodiment according to the invention, R¹⁰, R¹¹ and R¹² areidentical or different straight-chained C₁₋₆alkylene, arylene orarylene-C₁₋₆alkylene, and i is 0 or 1.

Alternatively, the spacer is a W-mer peptide, wherein W is an integerfrom 1 to 40.

In a particular embodiment, the substrates according to the inventionare selected from among:

The substrate according to the invention can be used in an assay whichis rapid, simple and easy to perform, subject to low variability, easilyautomated, and of low cost.

As the substrate of the subject invention provides for rapid and preciseassays, compared to assays described in the prior art, the assayaccording to the invention does not require measurement of the time thatis needed for absorbance to reach a certain value arbitrarilydetermined. Instead, assay time can be fixed at a shorter time comparedto prior art assays and the luminescence signal measured withtime-resolved luminometry. In a preferred embodiment said luminometry isfluorometry. Even at this shorter time, a more precise result isobtained. The end-point can, for example, refer to the time that isnecessary to reach a state in which about 80% of the substrate moleculesare cleaved by thrombin in the presence of an activator in a normalsample.

Alternatively, the end-point can also refer to the time that isnecessary to reach a steady state of the cleavage reaction by thrombinin a normal sample.

By washing off non-immobilised, thrombin-cleaved chelate-containingsubstrate fragments and non-immobilised, non-thrombin-cleaved substrate,the latter being present when the binding capacity of the immobilisationmatrix is less than the amount of substrate molecules, it is possible todirectly correlate the reduction of luminescence emission in the samplecompared to the luminescence from a thrombin-free standard sample(cleavage percentage) with the thrombin activity in the sample.

Alternatively, a substrate is immobilised onto the surface of animmobilisation matrix, such as a micro well or a micro particle.Sequentially, simultaneously or substantially simultaneously, a testsample, such as, for example, whole blood or plasma, together with anactivator, is added to the immobilisation matrix. The resulting reactionmixture is incubated to enable thrombin to act on the substrate, leadingto the release of thrombin-cleaved, chelate-containing substratefragments and leaving the binding partner-containing substrate fragmentand non-thrombin-cleaved intact substrate still on the immobilisationmatrix.

The identity of the activator used depends on the parameter PR, APTT orACT to be determined. Non-limiting examples of activators to be usedinclude thromboplastin, a partial thromboplastin reagent such as aphospholipid and contact activators such as silica, kaolin, celite,ellagic acid etc.

The amount of bioactive thrombin, i.e., thrombin not bound to anyinhibitor (e.g., antithrombin) and thus active in proteolytic activity,in the unknown sample can be determined from a standard curve forbioactive thrombin by its cleavage percentage (a decrease inluminescence intensity relative to total luminescence intensity).Subsequently, the level of bioactive thrombin can be expressed as theratio of bioactive thrombin from a group of normal samples (n≧20) tothat from an unknown sample. This ratio is equivalent to eitherprothrombin time ratio (PR), if the activator used is thromboplastin, oractivated partial thromboplastin time (APTT) ratio, if the activatorused is a partial thromboplastin reagent (e.g., a phospholipid) andcontact activator (e.g., silica, kaolin, celite, or ellagic acid), oractivated clotting time (ACT) ratio, if the activator used is a contactactivator and the sample type used is native whole blood.

In an embodiment of the invention, the addition of the test sample andactivator is performed simultaneously or substantially simultaneouslywith the addition of the substrate to an immobilisation matrix.

The one-step assay method without prior substrate immobilisation isshown schematically in FIG. 1. FIG. 1 is described in detail below.

This method is particularly useful when the substrate in question is noteasily cleavable when immobilised on the immobilisation matrix, but iscleavable by thrombin in the liquid phase, i.e., when the cleavagekinetics of the substrate in the liquid phase is faster than or at leastequally fast as the binding partner binding kinetics. Thus, both thecleavage reaction between the substrate and thrombin molecules and thebinding reaction between immobilised binding partner and cleaved as wellas intact substrate molecules take place during the following incubationstep.

In another embodiment of the invention, the addition of the test sampleis performed after immobilisation of the substrate to an immobilisationmatrix. The one-step assay method with prior substrate immobilisation isshown schematically in FIG. 2. FIG. 2 is described in detail below.

Such an assay has many advantages such as simplicity, rapidity androbustness. However, a condition thought to be a prerequisite for thisembodiment is that substrates should be cleavable when immobilised onthe immobilisation matrix.

Subsequently, non-immobilised, thrombin-cleaved chelate-containingsubstrate fragments are washed off. The luminescence emission from theimmobilised intact substrate is measured and compared to theluminescence from a thrombin-free standard sample. The percentage ofluminescence intensity reduction in the sample (cleavage percentage) isdirectly correlated with the thrombin activity in the sample.

The two-step assay can advantageously be utilised when the substrate inquestion performs well only in the liquid phase. The two-step assay isshown schematically in FIG. 3. FIG. 3 is described in detail below.

In this embodiment, a reaction mixture comprising a test sample, such asplasma or whole blood, an activator and a substrate is reacted in theliquid phase. Subsequently, part or all of said reaction mixture istransferred to an immobilisation matrix, where immobilisation ofthrombin-cleaved substrate fragments and non-thrombin-cleaved intactthrombin takes place. After washing off thrombin-cleavedchelate-containing substrate fragments and non-immobilised,non-thrombin-cleaved substrate, the level of luminescence fromimmobilised intact substrate is measured as above.

Which type of assay is chosen (such as a one-step or two-step assay)mainly depends on how difficult it is to perform a simultaneous additionof substrate, activator and sample to an immobilisation matrix. When itis difficult, a two-step assay is preferably used. When it is notdifficult, a one-step assay can be used. Besides, the two-step assay hasthe advantage over the one-step assay that the substrate amount appliedcan be very high as long as substrate solubility permits.

Another aspect of the present invention provides the use of a substrateaccording to the invention for determining the level of bioactivethrombin in a sample. The substrates according to the invention displayexcellent specificity for thrombin and can be quickly cleaved bythrombin, thus allowing the use of said substrates in an assay which iseasy to perform, which can be automated and which provides reliableresults in a short time.

In an embodiment, the present substrate is adopted specifically for thethrombin case, but it may as well be applied with different enzymessince the combination of a tri- or tetra-peptide (X) and the subjectlinker (Z) may be easily cleaved by other enzymes as well.

Thus, the substrate according to the present invention may be adapted tothe desired application by a minor modification of the substrate, morespecifically, by the choice of the peptide.

Different coagulation factors can be determined, such as active andinactive forms of FII, FV, FVII, FVIII, FIX, FX, FXI, and FXII, as wellas the active and inactive forms, fragments and combinations of C1, C2,C3, C4, C5, C6, C7, C8, and C9. Enzymes such as myeloperoxidase (MPO),alanine amino transferase (ALAT), aspartate amino transferase (ASAT),caspase-1 and glutathione peroxidase-1 (GPx-1) can also be determined.

In FIG. 1, a one-step assay without prior substrate immobilisation isshown schematically. The used substrate 1 comprises 4 parts ((1a, 1b, 1cand 1d) (A-X-Z-A′)), wherein 1a denotes the first partner of a bindingpair and 1a comprises optionally a spacer, 1b denotes a tri- ortetra-peptide, 1c denotes a linker and 1d denotes a luminescent chelate.2 denotes the second partner of the binding pair bound to the surface ofan immobilisation matrix 3. 4 denotes the presence of thrombin in thetest sample (A with a high level and B with a low level of thrombin) and5 denotes a thrombin free test sample (C).

In A, which represents a high level of thrombin, the substrate 1 reactswith the test sample 4. This results in a cleaved substrate. The cleavedsubstrate comprises two parts: a first part (1a, 1b) and a second part(1c, 1d). (1a, 1b) immobilises to the immobilisation matrix, while thesecond part (1c, 1d) of the substrate is washed away. Then, the emissionof luminescence from immobilised intact substrate (1a, 1b) is measuredand compared to the luminescence of a thrombin free sample 5 (C). Thewashing and measurement steps are not shown in FIG. 1.

FIG. 1 also illustrates the one-step assay with a low thrombin level(shown in B) and with a zero thrombin level (shown in C). C is used asthe control.

FIG. 2 illustrates a one-step assay as described above, but where theaddition of the test sample 4 or 5 is performed after immobilisation ofthe substrate 1 according to the invention to the immobilisation matrix3.

FIG. 3 illustrates a two-step assay. The first step (I) shows thatsubstrate 1 reacts with the test sample 4 (with thrombin (A)) or 5 (freeof thrombin (B)). This results in a thrombin cleaved substrate (A) or inan intact substrate (B). In step II, a part or all of the reactionmixture is transferred to an immobilisation matrix, where immobilisationof the thrombin-cleaved substrate fragment (1a, 1b) (A) andnon-thrombin-cleaved intact thrombin substrate 1 (1a, 1b, 1c and 1d) (B)takes place. After washing off the thrombin-cleaved chelate-containingsubstrate fragment (1c, 1d), the emission of luminescence from theimmobilised intact substrate 1 is measured as in the one step assay. Thewashing and measurements steps are not shown in FIG. 3.

Thus, with (A) a low level of chelate (luminescence/fluorescence) ismeasured, which represent a high level of thrombin. In contrast, with(B) a high level of chelate (luminescence/fluorescence) is measured,reflecting the absence of thrombin.

EXAMPLES

The present invention is described in more detail in the following,non-limiting specific examples.

For the below examples, the following materials and instrumentation wereused.

Materials

Sulfo-NHS-LC-LC-Biotin was purchased from Pierce, p-phenylenediamine waspurchased from Acros,(7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate(PyAOP) was purchased from Aldrich and N-ethyldiisopropylamine (DIPEA)was purchased from Fluka. Trifluoroacetic acid (TFA) was purchased fromRiedel-de Haën and triethylammonium acetate buffer (TEAA) was purchasedfrom Fluka. Acetonitrile (MeCN) was HPLC grade from J. T. Baker. DMF waspurchased from Lab-Scan and dried over molecular sieves (4 Å). All otherchemicals used were of analytical grade.

H-Phe(D)-Pip-Arg was purchased from KJ Ross-Petersen ApS.N-terminally-biotinylated long peptide-based D-Phe-L-Aze-Arg-OH such asBiotin-(Ser-(Gly)₂)₇ D-Phe-L-Aze-Arg-OH were purchased from Invitrogen.Innovin was purchased from Dade-Behring, Siemens. Bovine serum albumin(BSA) was purchased from Sigma.

9-dentate europium-chelate(2,2′,2″,2′″-{[2-(4-Isothiocyanatophenyl)-ethylimino]-bis(methylene)bis{4-{[4-(a-galactopyranoxy)phenyl]ethynyl}-pyridine-6,2-diyl}bis(methylenenitrilo)}tetrakis(acetato)europium(III)),wash solution and single cups coated with streptavidin were purchasedfrom Innotrac Diagnostics (Finland).

Low-fluorescence 12-well Maxisorp microtitration strips and singleMaxisorp microtitration wells (ultraviolet-quenched) were purchased fromNunc (Denmark).

Instrumentation

Mass spectrometry (MS) was operated on a Voyager DE-PRO (MALDI TOF) fromPerseptive Biosystems, using a-cyano-4-hydroxycinnamic acid as matrix. A1420 multilabel counter (Victor) from Wallac Oy, Perkin-Elmer lifeSciences was used to measure time-resolved fluorescence at 615 nm whenan assay was manually performed, while an Aio immunoanalyser fromInnotrac Diagnostics was used to measure time-resolved fluorescence at615 nm when an assay was semi-automatically performed.

Example 1 Synthesis of S1V6c

The synthesis of S1V6c involves three steps starting with the use of atripeptide comprising H-Phe(D)-Pip-Arg-OH as shown in FIG. 4. First, abiotin group with a linear spacer is connected to the N-terminalalpha-amino group of the tripeptide. Then, a diamine linker, such as4-phenylene-diamine, is coupled to the C-terminal carboxyl group of thetripeptide. Finally, a 9-dentate europium chelate is conjugated to thearomatic amino group of the introduced linker molecule.

Synthesis Procedure i) Biotinylation

H-Phe(D)-Pip-Arg-OH (5.0 mg, 11.6 μmol) was dissolved in PBS-buffer (pH7.0, 1 ml). Sulfo-NHS-LC-LC-Biotin (12.0 mg, 17.3 μmol) was added andthe mixture was stirred overnight at RT. The reaction mixture waspurified with HPLC. Yield: 5.5 mg (54%). MS: found 885.67. calculated885.15.

ii) Coupling of a Diamine-Linker

The biotinylated product from i) (2.4 mg, 2.7 μmol) was dissolved in dryDMF (0.5 ml). 4-Phenylene-diamine (1.2 mg, 10.84 μmol), PyAOP (1.7 mg,3.3 μmol) and DIPEA (0.6 μl, 3.3 μmol) were added and the mixture wasstirred overnight at RT. DMF was evaporated and the reaction mixture waspurified on a HPLC Column of Thermo ODS Hypersil (size: 150×10 mm). Alinear gradient made from A (0.1% TFA) and B (MeCN) in 20 minutes from5% B to 50% B was used for elution with a flow rate of 2.5 ml/min. Theyield was 2.4 mg (92%). MS found (975.83) was in close agreement with MScalculated (975.28).

iii) Labelling

The product from ii) (2.0 mg, 2.1 μmol) was dissolved in 50 mM carbonatebuffer (pH 9.8, 0.5 ml). 9-dentate europium-chelate (2,2′,2″,2′″-{[2-(4-Isothiocyanatophenyl)-ethylimino]-bis(methylene)bis{4-{[4-a-galactopyranoxy)phenyl]ethynyl}-pyridine-6,2-diyl}bis(methylenenitrilo)}tetrakis(acetato)europium(III))(3.3 mg, 2.5 μmol) was added and the reaction was stirred overnight atRT. The reaction mixture was purified on a HPLC Column of Phenomenex C18(size: 250×10 mm). A linear gradient made from A (20 mM TEAA) and B (20mM TEAA+50% MeCN) in 30 minutes from 5% B to 100% B was used for elutionwith a flow rate of 3 ml/min. The yield was 1.3 mg (27%).

Example 2 Synthesis of S1V12a

The synthesis of S1V12a involves only two steps (see FIG. 5) as thestarting material already contains a biotin group and a spacer groupmade of amino acids. After the same diamine linker, i.e.,4-phenylene-diamine is coupled to the C-terminal carboxyl group of thetripeptide, a 9-dentate europium chelate is conjugated to the aromaticamino group of the introduced diamine linker molecule. The experimentalconditions used for the synthesis of S1V12a were the same as those usedin the relevant steps for the synthesis of S1V6c.

Example 3

Three different reference standard curves to be used in subsequentthrombin determinations were constructed.

Standard curve A represents S1V6c in a concentration of 200 nM. Standardcurve B represents S1V9b in a concentration of 10 nM. Standard curve Crepresents S1V9b in a concentration of 25 mM. The substrates areprepared according to the conditions described in example 1.

Recombinant thromboplastin (Innovin) was used as an activator. The assaybuffer was a conventional HEPES buffer.

Standards of thrombin concentrations from 0 to 11.9 IU/ml were applied.

The assay was performed as a two-step assay. In step I, the thrombincontaining standards were added in different thrombin concentrations tothe assay buffer together with an activator and the substrate. Thereagents were mixed well and incubated at 37° C. for minutes.

In step II, a part of the reaction mixture was transferred to a microwell where the capture took place. The substrate was allowed toimmobilise/capture to the micro well for approximately 3 min at 37° C.before washing and drying. Non-captured substrate as well as the chelatecontaining cleaved substrate was washed away and the micro well was thendried. Finally fluorescence counts from the dried micro well weremeasured with a fluorescence counter.

The detected fluorescence counts were compared with a control samplefree of thrombin and for constitution of the standard curve, thereduction in signal compared to the reference (free of thrombin) wascalculated.

The results shown in FIG. 6, demonstrate that increasing concentrationsof S1V6c and S1V9b result in an increased reduction of signal. Thisdemonstrates that the substrate S1V6c and S1V9b are both very effectivesubstrates for thrombin. S1V9b resulted in a higher signal reduction atlower thrombin concentration compared to S1V6b. Still however, bothsubstrates are suitable thrombin substrates and both can therefore beused for determining the level of thrombin in a sample.

Example 4

For measurements in whole blood, the standard curve in example 3 may beapplied subject to the correction for the whole blood hematocrit value.Whole blood samples as well as plasma samples from 2 healthy female testpersons, were tested for thrombin according to the invention.

Four independent measurements on a plasma sample from test person one,showed a reduction in the detected signal compared to the control signalof 87.7%±0.5%. For test person two, the reduction was 91.5%±0.1%.

Similar results were obtained with whole blood samples, 80.7%±0.9% and86.3%±0.2%, respectively, for test person one and for test person two.

The results demonstrate the ability of the substrate and the assayaccording to the invention to measure thrombin in whole blood as well asin plasma samples and the invariability thereof.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the illustrativeexamples, make and utilize the present invention and practice theclaimed methods. All patents, patent applications and other referencescited throughout this application are herein incorporated by referencein their entirety.

1. A substrate for thrombin having the formula:A-X-Z-A′ wherein one of either A or A′ comprises a luminescent chelate,and the other one of A or A′ comprises a first partner of a bindingpair, optionally including a spacer, and connected via a peptide bond tothe remaining part of the substrate, X forms a tri- or tetra-peptideselected from among X′-Phe-Aze-Arg, X′-Phe-Pip-Arg, and X′-Phe-Pro-Arg,wherein X′ is absent or is selected from among Lys, Ahx, Ile, and Val, Zis NH—R-Z′, wherein R is selected from the group consisting ofC₁₋₆alkylene, C₁₋₆alkylenoxy, C₁₋₆thioalkylene, C₁₋₆thioalkylenoxy,carbonyl-C₁₋₆alkylene, carbonyl-C₁₋₆alkylenoxy, C₁₋₆alkylene-carbonyl,C₁₋₆alkylenoxy-carbonyl, C₁₋₆alkylene-arylene, C₁₋₆alkylenoxy-arylene,C₁₋₆alkylene-NH, C₁₋₆alkylenoxy-NH, C₁₋₆alkylene-NHCO,C₁₋₆alkylenoxy-NHCO, C₁₋₆alkylene-CONH, C₁₋₆alkylenoxy-CONH,C₁₋₆alkylene-COS, C₁₋₆alkylenoxy-COS,C₁₋₆alkylene-CONH—C₁₋₆alkylene-arylene, arylene, arylene-C₁₋₆alkylene,arylene-C₁₋₆alkylenoxy, R¹ _(a)-arylene-(NHCO—R²)_(b), R³_(c)-arylene-(CONH—R⁴)_(d), (R⁵—CONH)_(e)-arylene-R⁶ _(f), and(R⁷—NHCO)_(g)-arylene-R⁸ _(h), wherein each R¹, R², R³, R⁴, R⁵, R⁶, R⁷,and R⁸ at each occurrence is independently selected from amongC₁₋₆alkylene, C₁₋₆alkylenoxy, C₁₋₆thioalkylene, C₁₋₆thioalkylenoxy,carbonyl-C₁₋₆alkylene, carbonyl-C₁₋₆alkylenoxy, C₁₋₆alkylene-carbonyl,arylene and arylene-C₁₋₆alkylene, and each of a, b, c, d, e, f, g, and his independently selected from among the integers from 0 to 6, whereinthe arylene is phenylene, biphenylene or naphthylene, which phenylene,biphenylene or naphthylene is optionally mono-, di- or tri-substitutedby one or more substituents selected from among halogen, OH, SH, CN,NO₂, C₁₋₆alkyl, C₁₋₆alkoxy, and C₁₋₆alkoxycarbonyl, Z′ comprises one ofN, S, and carbonyl, wherein, when Z′ comprises N, Z′ is selected fromamong thiourea (—NH—CS—NH—), aminoacetamide (—NH—CO—CH₂—NH—), amide(—NH—CO—), methylamide (—NCH₃—CO—) and substituted-triazine-diamine(—NH—(R⁹C₃N₃)—NH—), when Z′ comprises S, Z′ is selected from amongthioether (—S—), thioacetamide (—S—CH₂—CO—NH—), disulfide (—S—S—),(—S—CO—CH₂—NH—) and (—S—(R⁹C₃N₃)—NH—) or when Z′ comprises carbonyl, Z′is selected from among an amide (—CO—NH—, —CO—NCH₃—) and an ester(—CO—O—), wherein R⁹ is selected from the group consisting of hydrogen,halogen, C₁₋₆alkyl, C₁₋₆thioalkyl, C₁₋₆alkoxy, C₁₋₆thioalkoxy, aryloxy,and amino, which alkyl, thioalkyl, alkoxy, thioalkoxy or aryloxy groupis optionally mono-, di- or tri-substituted and which amino group isoptionally mono- or di-substituted by one or more substituents selectedfrom among halogen, OH, SH, CN, NO₂, C₁₋₆alkyl, C₁₋₆alkoxy, andC₁₋₆alkoxycarbonyl.
 2. The substrate according to claim 1, wherein X isD-Phe-L-2-Aze-L-Arg or D-Phe-L-2-Pip-L-Arg.
 3. The substrate accordingto claim 1, wherein Z′ is N and R is arylene, C₁₋₆alkylene-arylene, orR¹ _(a)-arylene-(NHCO—R²)_(b), wherein a and b are independently 0, 1 or2.
 4. The substrate according to claim 1, wherein said luminescentchelate is a fluorescent lanthanide chelate.
 5. The substrate accordingto claim 4, wherein said lanthanide chelate is selected from among


6. The substrate according to claim 1, wherein said first partner of abinding pair is selected from among biotin/streptavidin, biotin/avidin,biotin/biotin acceptor peptide, and streptavidin derivative/acceptorpeptide.
 7. The substrate according to claim 1, characterised in thatsaid first partner of a binding pair is connected to the remaining partof the substrate via a spacer of the formulaNH—R¹⁰—CONH—R¹¹—CO—(NH—R¹²—NH)_(i), wherein each of R¹⁰, R¹¹ and R¹² isindependently selected from among C₁₋₆alkylene, C₁₋₆alkylenoxy,C₁₋₆thioalkylene, C₁₋₆thioalkylenoxy, carbonyl-C₁₋₆alkylene,carbonyl-C₁₋₆alkylenoxy, C₁₋₆alkylene-carbonyl, arylene andarylene-C₁₋₆alkylene, wherein the arylene is phenylene, biphenylene ornaphthylene, which phenylene, biphenylene or naphthylene is optionallymono-, di- or tri-substituted by one or more substituents selected fromamong halogen, OH, SH, CN, NO₂, C₁₋₆alkyl, C₁₋₆alkoxy, andC₁₋₆alkoxycarbonyl, and wherein i is an integer from 0 to 6, or thespacer is a W-mer peptide, wherein W is an integer from 1 to
 40. 8. Thesubstrate according to claim 7, wherein R¹⁰, R¹¹ and R¹² are identicalor different straight-chained C₁₋₆alkylene, arylene orarylene-C₁₋₆alkylene and i is 0 or
 1. 9. The substrate according toclaim 1 selected from among S1V6a, S1V6b, S1V6c, S1V8, S1V9a, S1V9b,S1V12a, S1V12b and S1V12c


10. A one-step assay method for determining the level of bioactivethrombin in a test sample comprising the steps: a) combining,sequentially, simultaneously or substantially simultaneously, asubstrate according to claim 1, an activator and said test sample; b)incubating the resulting reaction mixture to release thrombin-cleaved,chelate-containing substrate fragments and immobilising the bindingpartner-containing substrate fragment and non-thrombin-cleaved intactsubstrate on an immobilisation matrix; c) washing off non-immobilised,thrombin-cleaved chelate-containing substrate fragment andnon-immobilised, non-thrombin-cleaved substrate, if present; d)measuring the level of luminescent emission from immobilized intactsubstrate; and e) calculating thrombin activity from the reduction ofintensity of luminescent emission compared to a thrombin-free standardsample.
 11. The assay method according to claim 10, wherein the additionof said sample and activator is performed simultaneously orsubstantially simultaneously with the addition of the substrate to animmobilisation matrix.
 12. The assay method according to claim 10,wherein the addition of said sample and activator is performed afterimmobilisation of the substrate on an immobilisation matrix.
 13. Atwo-step assay method for determining the level of bioactive thrombin ina test sample comprising the steps: a) adding said sample and anactivator to a substrate according to claim 1 in liquid phase, b)incubating the resulting reaction mixture to release thrombin-cleaved,chelate-containing substrate fragment, c) adding the reaction mixture toan immobilisation matrix, d) washing off non-immobilised,thrombin-cleaved chelate-containing substrate fragment andnon-immobilised, non-thrombin-cleaved substrate, if present, e)measuring the level of luminescent emission from immobilised intactsubstrate, and f) calculating thrombin activity from the reduction ofintensity of luminescent emission compared to a thrombin-free standardsample.
 14. The assay method according to any of claims 10-13, whereinsaid activator is selected from the group consisting of thromboplastin,partial thromboplastin reagents and contact activators.
 15. The assaymethod according to claim 14, wherein said partial thromboplastinreagents comprise phospholipids, and said contact activators comprisesilica, kaolin, celite or ellagic acid.
 16. A test kit for determiningthe level of bioactive thrombin in a sample comprising a) a luminescentsubstrate according to claim 1, and b) a solid immobilisation matrix.17. A substrate for an enzyme having the formula:A-X-Z-A′ wherein one of either A or A′ comprises a luminescent chelate,and the other one of A or A′ comprises a first partner of a bindingpair, optionally including a spacer, and connected via a peptide bond tothe remaining part of the substrate, X forms a tri- or tetra-peptide, Zis NH—R-Z′, wherein R is selected from the group consisting ofC₁₋₆alkylene, C₁₋₆alkylenoxy, C₁₋₆thioalkylene, C₁₋₆thioalkylenoxy,carbonyl-C₁₋₆alkylene, carbonyl-C₁₋₆alkylenoxy, C₁₋₆alkylene-carbonyl,C₁₋₆alkylenoxy-carbonyl, C₁₋₆alkylene-arylene, C₁₋₆alkylenoxy-arylene,C₁₋₆alkylene-NH, C₁₋₆alkylenoxy-NH, C₁₋₆alkylene-NHCO,C₁₋₆alkylenoxy-NHCO, C₁₋₆alkylene-CONH, C₁₋₆alkylenoxy-CONH,C₁₋₆alkylene-COS, C₁₋₆alkylenoxy-COS,C₁₋₆alkylene-CONH—C₁₋₆alkylene-arylene, arylene, arylene-C₁₋₆alkylene,arylene-C₁₋₆alkylenoxy, R¹ _(a)-arylene-(NHCO—R²)_(b), R³_(c)-arylene-(CONH—R⁴)_(d), (R⁵—CONH)_(e)-arylene-R⁶ _(f), and(R⁷—NHCO)_(g)-arylene-R⁸ _(h), wherein each R¹, R², R³, R⁴, R⁵, R⁶, R⁷,and R⁸ at each occurrence is independently selected from amongC₁₋₆alkylene, C₁₋₆alkylenoxy, C₁₋₆thioalkylene, C₁₋₆thioalkylenoxy,carbonyl-C₁₋₆alkylene, carbonyl-C₁₋₆alkylenoxy, C₁₋₆alkylene-carbonyl,arylene and arylene-C₁₋₆alkylene, and each of a, b, c, d, e, f, g, and his independently selected from among the integers from 0 to 6, whereinthe arylene is phenylene, biphenylene or naphthylene, which phenylene,biphenylene or naphthylene is optionally mono-, di- or tri-substitutedby one or more substituents selected from among halogen, OH, SH, CN,NO₂, C₁₋₆alkyl, C₁₋₆alkoxy, and C₁₋₆alkoxycarbonyl, Z′ comprises one ofN, S, and carbonyl, wherein, when Z′ comprises N, Z′ is selected fromamong thiourea (—NH—CS—NH—), aminoacetamide (—NH—CO—CH₂—NH—), amide(—NH—CO—), methylamide (—NCH₃—CO—) and substituted-triazine-diamine(—NH—(R⁹C₃N₃)—NH—), when Z′ comprises S, Z′ is selected from amongthioether (—S—), thioacetamide (—S—CH₂—CO—NH—), disulfide (—S—S—),(—S—CO—CH₂—NH—) and (—S—(R⁹C₃N₃)—NH) or when Z′ comprises carbonyl, Z′is selected from among an amide (—CO—NH—, —CO—NCH₃—) and an ester(—CO—O—), wherein R⁹ is selected from the group consisting of hydrogen,halogen, C₁₋₆alkyl, C₁₋₆thioalkyl, C₁₋₆alkoxy, C₁₋₆thioalkoxy, aryloxy,and amino, which alkyl, thioalkyl, alkoxy, thioalkoxy or aryloxy groupis optionally mono-, di- or tri-substituted and which amino group isoptionally mono- or di-substituted, by one or more substituents selectedfrom among halogen, OH, SH, CN, NO₂, C₁₋₆alkyl, C₁₋₆alkoxy, andC₁₋₆alkoxycarbonyl.